3D Structure and Function of the Mammalian Kinetochore

在真核细胞分裂中，子染色体分配到子细胞中是通过将染色体附着到微管上来实现的，微管然后在胞质分裂之前将染色体分开。微管附着在染色体上的部位是一个在形态学和生物化学上都很独特的染色体区域，称为动粒。 麦克尤恩博士最近的工作采用了最先进的保存和高分辨率成像技术，挑战了过去三十年来在该领域已被接受的动粒的“三层”形态模型，并且由于传统固定方案中固有的固定伪影，该模型可能不准确。 现在，麦克尤恩博士的长期目标是确定哺乳动物动粒的高分辨率结构图，以解释和预测功能机制。该研究将光学和电子显微镜的最先进方法与分子方法相结合，以计算结构图并获得关键动粒组分的功能表征。本项目有以下四个目标：研究目标1.建立哺乳动物动粒的原型高分辨率3D结构图。1a：确定动粒的关键特征，并寻找亚基结构的证据。1b.检验以下假设：动粒具有离散的结构域，这些结构域动态地响应微管的附着和剥夺，并且扩大的外部结构域与混杂的微管捕获和高非整倍体发生率相关。研究目的2.测试关于CENP-E在有丝分裂中的分子作用的三个假设。2a：CENP-E在维持动粒微管附着的稳定性中起作用。2b：CENP-E防止单取向染色体过度向极移动进入纺锤体极。2c：CENP-E不需要用于后期开始检查点的释放。验证CENP-E与ZW 10/动力蛋白在结合动粒微管中的功能是冗余的，并且在向极运动中对抗动力蛋白的假设。测试的假设，动粒是顺序组装，与动粒蛋白CENP-C和BUB 1需要完整的组装。这项研究项目将提供重要的和有意义的新信息的复杂性动粒结构和功能。 它将填补我们目前关于动粒分子组分如何排列以及它们如何与细胞分裂装置的其他组分相互作用的知识中的一个主要空白。

In eukaryotic cell division, the partitioning of daughter chromosomes into the daughter cells is achieved by attachment of the chromosomes to microtubules which then move the chromosomes apart prior to cytokinesis. The site of attachment of the microtubules to the chromosomes is a morphologically and biochemically distinctive chromosomal region called the kinetochore. Recent work by Dr. McEwen, employing state-of-the-art preservation and high resolution imaging technologies, has challenged the "trilaminar" morphological model of the kinetochore that has been accepted in the field for the past thiry years, and which was probably inaccurate due to fixation artifacts inherent in conventional fixation protocols. Now,Dr. McEwen's long-range goal is to define a high-resolution structural map of the mammalian kinetochore that explains and predicts functional mechanisms. The research combines state-of-the-art methods of light and electron microscopy with molecular approaches to compute the structural map and obtain functional characterization of key kinetochore components. The current project has four objectives, listed below.Research Objective 1. Establish a prototype high-resolution 3D structural map of the mammalian kinetochore. 1a: Identify key features of the kinetochore and look for evidence of a subunit structure. 1b. Test the hypothesis that the kinetochore has discrete structural domains that dynamically respond to microtubule attachment and deprivation, and that an enlarged outer domain is correlated with promiscuous microtubule capture and a high incidence of aneuploidy.Research Objective 2. Test three hypotheses concerning the molecular role of CENP-E in mitosis. 2a: CENP-E has a role in maintaining the stability of kinetochore microtubule attachments. 2b: CENP-E prevents monooriented chromosomes from over-traveling poleward into the spindle pole. 2c: CENP-E is not required for release of the anaphase onset checkpoint.Research objective 3. Test the hypotheses that CENP-E function is redundant with ZW10/dynein in binding kinetochore microtubules, and opposes dynein in poleward motion.Research objective 4. Test the hypothesis that the kinetochore is sequentially assembled, with kinetochore proteins CENP-C and BUB1 being required for complete assembly.This research project will provide important and significant new information on the complexities of kinetochore structure and function. It will fill a major gap in our present knowledge of how molecular components of the kinetochore are arranged and how they interact with other components of the cell division apparatus.